For researchers starting out in the field of miRNA research, the miScript PCR Starter Kit provides all the necessary components for set-up of miRNA quantification experiments. The kit includes reagents for reverse transcription and real-time PCR, a miScript PCR Control for RNU6-2 for normalization, and a miScript Primer Assay for detection of human miR-15a.

The miScript PCR Starter Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Accurate, linear detection of miR-15a.

RNA was purified from HeLa S3 cells. Using the miScript PCR Starter Kit, cDNA was generated and a range of cDNA amounts from 10 pg to 10 ng was used in real-time PCR to detect miR-15a. Real-time PCR was performed in the Rotor-Gene Q cycler. The resultant CT values decreased linearly with increasing cDNA amount, indicating quantitative detection from a wide range of template amounts.

Performance

miR-15a is highly expressed in human tissues and cells, making it ideal for use as a positive control. The miScript PCR Starter Kit enables accurate, linear detection of miR-15a in positive control experiments for setup and optimization of miRNA quantification (see figure Accurate, linear detection of miR-15a).

Principle

The miScript PCR System enables easy miRNA quantification and profiling using SYBR® Green-based real-time PCR. The miScript PCR System covers all the steps of conversion of RNA into cDNA to detection of miRNAs in SYBR® Green-based real-time PCR. The miScript PCR Starter Kit combines the key components of the miScript PCR System in a single kit, allowing the user to establish miR-15a quantification and normalization of results, before proceeding to miRNA quantification in their field of interest.

Procedure

cDNA prepared in a reverse transcription reaction using miScript HiSpec Buffer or miScript HiFlex Buffer serves as the template for real-time PCR. Real-time PCR is performed using a human miR-15a-specific miScript Primer Assay (forward primer) and the miScript Universal Primer (reverse primer) with QuantiTect SYBR Green PCR Master Mix. For normalization, a parallel reaction is performed using the human RNU6-2 (RNU6B) miScript Primer Assay and the miScript Universal Primer with QuantiTect SYBR Green PCR Master Mix.